Bacterial endotoxins are lipopolysaccharide (LPS) molecules found in the outer membrane of the Gram-negative bacterial cell wall. Their known toxic biological activities include pyrogenicity, leukopenia and leukocytosis, provocation of disseminated intravascular coagulation, and depression of blood pressure. Endotoxins may contribute significantly to the morbidity and mortality of Gram-negative sepsis, yet other endotoxin activities (such as induction of tumor necrosis, adjuvance, and B-cell mitogenicity) may have beneficial value for the host. Careful definition of the role of endotoxins in human infection and immunity will require reliable assays for these molecules in plasma. This project will evaluate two approaches to the problem of endotoxin assay. The proposed immunoassay is a modification of the enzyme-linked immunoadsorbant technique, in which LPS will act as the "filling" for a "sandwich" made of solid-phase antibody and antibody-enzyme complex. This assay, which should give a measure of the amount of endotoxin present in a sample, will be adapted to the quantitation of both specific (O) and common (lipid A, R-core) regions of the LPS molecule. The second approach will be a modification of the Limulus lysate, a test of endotoxin activity which has previously been too non-specific and semi-quantitative for routine measurement of endotoxin levels in plasma. Endotoxin activates an enzyme in the lysate. The activated enzyme cleaves a substrate protein, and the resulting fragments polymerize to form a gel. Polymerization of radiolabeled substrate monomers will be measured during the early stages of the gelation reaction. It is hoped that this quantitative approach will provide greater sensitivity than has previously been possible with the limulus assay. Improved specificity may be achieved using immunoadsorption to remove LPS from control specimens. These assays will be evaluated in models of focal and bacteremic Gram-negative infection in experimental animals.